Gene delivery using non-viral (plasmid) techniques are very desirable due to their economic, convenience, ease of manufacturing, cost-effectiveness, and safe characteristics. The techniques are currently limited by low transfection efficiencies. To address this limiting factor, our research focuses on developing nano-sized calcium phosphate (CAP) particles called "NanoCaPs" as a novel delivery agent for plasmid DNA transfection. The reduced crystallite dimensions of nano-sized materials, compared to conventionally synthesized micron sized CaP will result in increased surface-active DNA binding sites and enhanced reactivity, thus improving transfection efficiencies. Furthermore, the NanoCaPs are biocompatible and they could be used alone or incorporated in many synthetic or natural polymers to deliver genes in a sustained manner thus providing an elegant plasmid gene delivery system. The NanoCaPs have the potential to provide nucleic-acid based therapeutics that closely resembles traditional pharmaceuticals and gene delivery for tissue engineering. The overall objective of this grant application is to engineer a safe and versatile plasmid gene delivery system for tissue engineering applications. Our goal is to demonstrate that the synthesized NanoCaPs incorporated in a natural (fibrin, collagen etc.) or synthetic (PLA, PLGA, PCL, polyurethane etc.) biomimetic extracellular matrix (bECM) will enhance both the in vitro and in vivo transfection efficiency of plasmid DNA (pDNA) by increasing the uptake and expression of marker genes (Luciferase and/or LacZ). This will be achieved using quantitative digital imaging methods. Our preliminary in vitro data is in excellent agreement with our objective to design and develop an efficient plasmid gene therapy that could be utilized with or without biodegradable polymers for gene replacement therapy and tissue engineering. The present study will provide a concrete foundation for conducting further applied and basic science research related to plasmid gene therapy. The specific aims have been formulated to provide solutions to fundamental questions related to in vitro and in vivo pDNA transfection efficiency and the application of the pDNA/NanoCaPs/polymer in bone tissue engineering thus providing key information that is currently not available. The proposed research will enable the generation of novel carriers for plasmid gene delivery, the benefits of which will be seen in both hard and soft tissue engineering applications.